It has long been established that b-cells within whole islets secrete more insulin than do isolated b-cells. This functional enhancement may arise from gap junctional coupling between cells in the intact islet. Until now, the behavior of b-cells within islets has been largely inferred from in vitro studies of dispersed cells and biochemical analysis of whole islets. Dr. Piston has begun to study the function of individual b- cells within intact islets by optimizing recent advances in quantitative optical imaging for this purpose. This has led to novel experiments which show marked differences between isolated b-cells and whole islets in NAD(P)H autofluoresence (an endogenous reporter of intracellular metabolism) and glucokinase (GK) expression. These results demonstrate a new level of biological complexity that can be investigated only by detailed studies of intact islets. Dr. Piston hypothesizes that insulin secretion is directly coupled to glucose metabolism, and that while both responses are rate-limited by GK activity under normal physiological conditions, this rate-limiting role may be lost by only a slightly over-expression of GK. The applicant also hypothesizes that gap junctional coupling between b- cells in the islet accounts for increased insulin secretion from b- cells in the intact islet over isolated b- cells. The validity and limits of these hypotheses will be addressed through three specific aims: 1) To determine the temporal relationship between the magnitude of glucose-stimulated NAD(P)H response and amount of insulin secretion; 2) To determine the range of expression levels over which GK maintains its rate-limiting role in islet metabolism and secretion; 3) To determine the ability of intercellular coupling to enhance insulin secretion. The proposed experiments take advantage of the applicant's demonstrated expertise in two-photon excitation and confocal microscopic techniques, combined with several available transgenic mouse models. The applicant concludes that these investigations will advance our understanding of the in vivo interplay of biochemical mechanisms known to be involved in glucose-stimulated insulin secretion from pancreatic islets.